Imaging system

ABSTRACT

An imaging system is described for measuring the position or movement of a particle having a size of less than about 20 microns. The system comprises an optional sample holder configured to hold a sample with a particle, an optional illumination source configured to illuminate the sample, a lens having a magnification ratio from about 1:5 to about 5:1 and configured to generate the image of the sample, an image sensor having a pixel size of up to about 20 microns and configured to sense the image of the sample, and an image processor operatively connected to the image sensor to process the image of the particle in order to determine the position or movement of the particle. The dimension of the image of each particle is at least about 1.5 times the dimension of the particle multiplied by the magnification ratio of the lens, and the image of each particle is distributed on at least two pixels of the sensor. The imaged area of the sample is at least about one millimeter squared.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority, as acontinuation application under 35 U.S.C. § 120, of U.S. Ser. No.15/444,462, entitled IMAGING SYSTEM and filed on Feb. 28, 2017, to Xu,et al., which in turn claims the benefit under 35 U.S.C. § 119(e), ofU.S. provisional patent application Ser. No. 62/352,222 filed Jun. 20,2016, the contents of which applications are incorporated in theirentirety herein by reference, for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.1R43AI122527-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention is generally in the field of imaging techniques andrelates to an imaging device and system to measure the position ormovement of small particles in a sample.

BACKGROUND OF THE INVENTION

Measuring the position and movement of small particles is essential inmany scientific fields. Typical examples are microbead tracking and celltracking in biomedical research. Microbeads are uniform polymerparticles of about 0.3 to 500 microns in diameter, which can be used toabsorb or couple with bio-reactive molecules such as proteins or nucleicacids. Microbeads are used to track the position of biomolecules insingle molecule manipulation experiments. In such an application,microbeads are pre-coupled with a ligand, a biomolecule such as anantibody, streptavidin, protein, antigen, DNA/RNA or other molecule,which binds to the desired target molecule. The whole assembly can bemanipulated precisely with a magnetic or optical field. An example ofcell tracking is the sperm motility test, in which the count, shape, andmovement of sperm cells are measured to evaluate the health status ofthe sperm, in order to provide information for infertility treatment.

In these applications, high accuracy of the position measurement of theparticles and large field of view are two desirable specifications. Theaccurate position of the investigated particles can be computed toderive precisely the movement, trajectory, speed, acceleration or otherbehavior of the particles. The large field of view enables a largenumber of particles to be characterized, which can significantly enhancethe statistics, precision, sensitivity, and certainty of the system. Forinstance, if thousands or tens of thousands of sperm can becharacterized simultaneously in one motility test, more preciseconclusions can be drawn from the evaluation.

Unfortunately, the concepts of high accuracy of the position measurementof the particles and large field of view often conflict in traditionalimaging systems. To accurately calculate the position or movement of theparticle, a magnified image of the particle is preferred. The larger theimage of the particle, and the more pixels the image covers in the imagesensor, the more precise can the position of the particle be calculated.However, the use of high magnification reduces the field of view to thesize of the image sensor divided by the magnification ratio.

A lens-free technique is described in US20120248292A1 to obtain a largefield of view with high-resolution image of small particles, but theinvestigated sample has to be located on the surface of the image sensorwithout any gap. As a result, the image sensor has to be replaced ineach test, which is very costly and the imaging system is difficult toconfigure. For instance, most commercially available image sensors havea transparent window enclosing the sensor chip, and this window has tobe removed to locate the sample at the surface of the chip. What isneeded then is a system which is easy to build without the need tolocate the sample adjacent to the image sensor, to obtain bothhigh-accuracy position measurement of small particles and a large fieldof view.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an imaging system fordetermining the position or movement of a particle in a sample,comprising:

a. an optional sample holder configured to hold a sample containing aparticle;

b. an optional illumination source configured to illuminate the sample;

c. a lens having a magnification ratio of from about 1:5 to about 5:1and configured to generate an image of the particle in the sample,wherein the dimension of the image of the particle is at least about 1.5times the dimension of the particle multiplied by the magnificationratio of the lens, and wherein the size of the particle is less thanabout 20 microns, and wherein the imaged area of the sample is at leastabout one mm²;

d. an image sensor having a pixel size of less than about 20 microns andconfigured to sense the image of the particle, wherein the image of theparticle is distributed on at least two pixels; and

e. an image processor operatively connected to the image sensor toprocess the image of the particle in order to determine the position ormovement of the particle.

In another aspect, the present invention provides a method ofdetermining the position or movement of a particle in a sample,comprising the steps of:

a. providing a sample having a particle, wherein the size of theparticle is less than about 20 microns;

b. optionally illuminating the sample;

c. generating an image of the particle with a lens having amagnification ratio of from about 1:5 to about 5:1, wherein thedimension of the image of the particle is at least about 1.5 times thedimension of the particle multiplied by the magnification ratio of thelens, and wherein the imaged area of the sample is at least about 1 mm²;

d. sensing the image of the particle on at least two pixels of an imagesensor having a pixel size of less than about 20 microns; and

e. processing the image of the sample to measure the position ormovement of the particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representative view of a sample with small particles.

FIG. 1B is a representative view of the images of the small particles onan image sensor. The actual size of the particle is smaller than thepixel size of the sensor. The dimension of the image of each particle islarger than the actual dimension of the particle multiplied by themagnification ratio of the lens because of the blurring effect caused bythe low optical resolution of the imaging system.

FIG. 1C depicts the intensity of the blurred image of a small particledistributed on multiple pixels of a sensor when low optical resolutionis applied. The intensity distribution is only shown in one dimension. AGaussian profile is used to fit the intensity distribution, in order topermit precise measurement of the particle position.

FIG. 1D depicts the intensity of the blurred image of a small particledistributed on multiple pixels of the sensor when the particle moves adistance of about ⅓ of the pixel size from its initial position depictedin FIG. 1C. The intensity distribution is only shown in one dimension.Using a Gaussian profile to fit the intensity distribution candistinguish the movement of the particle with a high degree ofprecision.

FIG. 1E depicts the intensity of the sharp image of a small particledistributed on one pixel of the sensor when high optical resolution isapplied as in a traditional imaging system. The intensity distributionis only shown in one dimension. Because the intensity of the image ofthe particle is only located at one pixel, the precision of the positioncannot be higher than the pixel size.

FIG. 1F depicts the intensity of the sharp image of a small particledistributed on one pixel of the sensor when the particle moves adistance of about ⅓ of the pixel size from its initial position depictedin in FIG. 1E. The intensity distribution is only shown in onedimension. Because the image of the particle is still located at thesame single pixel, the movement of this particle cannot be detected.

FIG. 2 is a schematic representation of one embodiment of the imagingsystem (30). The system comprises a sample holder (31) to hold a samplewith small particles, an illumination source (32) to illuminate thesample, a lens (33) to generate an image of the sample, an image sensor(34) to sense the image of the sample, and an image processor (35) toprocess the image or images of the sample to measure the position ormovement the particles, and an optional cable (36) connecting the imagesensor (34) and the image processor (35).

FIG. 3 is a schematic representation of another embodiment of theimaging system (40). The system comprises an optional sample holder (41)to hold a sample with small particles, an optional illumination source(42) to illuminate the sample, a reflecting mirror (43) to fold theoptical path, a lens (44) to generate the image of the sample, an imagesensor (45) to sense the image of the sample, and an image processor(46) to process the image or images of the sample to measure theposition or movement the particles, and an optional cable connecting(47) the image sensor (45) and the image processor (46).

FIG. 4A depicts the displacement of a bead using an embodiment of thepresent invention. The bead (54) was hybridized with the first probe(52), the target (51), and the second probe (53) and tethered to theglass surface coated with protein (55). A first position for each beadwas determined in images taken when the flow runs in one direction, andthen a second position for each bead was determined in images taken whenthe flow runs in the opposite direction. Bead displacement is thedistance between the first and second positions.

FIG. 4B shows histograms of bead displacement generated using anembodiment of the present invention, with the target concentration as400 femtomolar (left), 100 femtomolar (middle) and 0 femtomolar (right).Beads that are displaced more than 5.8 microns by the flow indicatetarget presence, and form the peak on the right (57) in each histogram.Beads that are displaced less than 5.8 microns form a peak on the left(58) and correspond to beads attached to the glass via a non-specificinteraction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a system that is capable of measuring theposition or movement of small particles over a large area with a highdegree of accuracy. Preferably, the particle size is less than about 20microns, more preferably from about 0.5 microns to about 2 microns. Incertain embodiments, the magnification of the imaging system is equal toor smaller than about 1:1, which provides that the field of view is atleast the size of the image sensor. In addition, the numeric aperture ofthe lens is sufficiently small, and the imaging system has asufficiently low optical resolution, to result in a blurred image ofeach individual particle. In this instance, the size of the blurredimage is larger than the dimension of the particle multiplied by themagnification ratio of the imaging system. Further, in certainembodiments an image sensor with a small pixel size is used, whichresults in the intensity of the image being distributed on multiplepixels. The signals from these multiple pixels are analyzed to obtainaccurate position or movement of each particle. The present inventionthus allows for precise positioning of a large number of small particlesin a large field of view.

Field of view is the area of the object under inspection that an imagingsystem can acquire. In a preferred embodiment, the field of view willencompass from about 100 to about 1 million particles in the imaged areaof the sample.

A lens is a transmissive optical device that affects the focus of alight beam through refraction, so it can focus light to form an image.Lenses are made from transparent materials such as glass, ground andpolished to a desired shape.

The magnification ratio of a lens is the ratio of the apparent size ofan object (or its size in an image) to its true size, and thus it is adimensionless number.

The numerical aperture (NA) of a lens is a dimensionless number thatcharacterizes the range of angles over which the lens can accept light.Mathematically, NA is equal to n*sin 0, where n is the index ofrefraction of the lens medium, and 0 is the maximal half-angle of thecone of light that can enter the lens.

The optical resolution of an imaging system describes its ability toresolve detail in the object that is being imaged, which is defined bythe distance between two distinguishable radiating points.Mathematically, optical resolution is equal to 0.61λNA, where λ is thelight wavelength and NA is the numerical aperture of the lens. Ingeneral, high optical resolution results in a sharp image with moredetails distinguished, and low optical resolution leads to a blurredimage.

In a preferred embodiment, a lens with a sufficiently small numericalaperture is employed to make the optical resolution sufficiently low,which results in blurred images of the small particles.

Referring now to the Figures, FIG. 1A represents an area in the samplecontaining five small particles, including particle 1. FIG. 1Brepresents the image of the sample formed at the image sensor 12 with1:1 magnification. The image comprises multiple pixels (one of which isshown filled in at 13). In FIG. 1A, virtual grid 2 shows the scale ofthe sample, and one square in the virtual grid has the same size of onepixel in the sensor according to 1:1 magnification. The actual size ofthe particle is smaller than the pixel size of the sensor. Particle 1 islocated at approximately the center of its corresponding grid unit. InFIG. 1B, image 11 is the image of the small particle 1, and the size ofimage 11 is larger than the actual dimension of particle 1 multiplied bythe magnification ratio of the lens (equal to 1 in the depicted case)because of the blurring effect. Image sensor 12 has a sufficiently smallpixel size such that the intensity of the blurred image 11 isdistributed on multiple pixels. Preferably, the pixel size is less thanabout 20 microns, more preferably less than about 5 microns.

Based on the intensity profile distributed on these multiple pixels, theposition of particle 1 can be calculated with high precision. Forexample, fitting a Gaussian model of the profile (21 in FIG. 1C) to fitthe actual intensity of the image of the particle, can be utilized tocalculate the position of the particle (22 in FIG. 1C) more precisely.FIG. 1D shows the position measurement when particle 1 moves a distanceof about ⅓ of the pixel size from its initial position in FIG. 1C, andits new position (25 in FIG. 1D) can be measured with high precision.The intensity distribution is only shown in one dimension in FIG. 1C andFIG. 1D. Therefore, imaging with low optical resolution that results inblurred particle images allows for high precision (sub-pixel) particleposition measurements.

Instead, FIG. 1E and FIG. 1F show the position measurement when highoptical resolution is used as in conventional imaging systems. Highoptical resolution results in the image size of particle 1 being equalto the actual dimension of particle 1 multiplied by the magnificationratio of the lens (equal to 1 in this case), and thus the intensity ofthe image of the particle is only located within one pixel. As a result,the precision of its position cannot be better than one pixel size. Whenthe particle moves a distance of about ⅓ of the pixel size, thismovement cannot be detected because the image of the particle is stilllocated within the same single pixel.

The blurring effect caused by low optical resolution is advantageous inthis invention. The advantage of this invention is demonstrated in FIG.1—with 1:1 magnification, the field of view is kept as the same size ofthe image sensor without any compromise, but the image of the particleis enlarged to help measure the position of the particle more precisely.

FIG. 2 depicts one preferred configuration of the imaging system. Thesample holder 31 contains the sample to be imaged, and small particlesare present in the sample. The precise structure of the sample holder isnot critical, and could include, e.g., a capillary or glass slide.Illumination source 32 is used to illuminate the sample, if needed. Lens33 is configured to generate an image of the sample. Sensor 34 isconfigured to sense the image of the sample generated by lens 33. Imageprocessing unit 35 processes the image or images of the sample todetermine the position or movement the particles. In this figure, theimage processing unit is depicted as a laptop computer, but one ofordinary skill would recognize that many other computing devices couldbe utilized, such as a desktop computer, a tablet computer, asmartphone, an embedded processor in an embedded system, etc. Optionalcable 36 connects the image sensor 34 and the image processor 35;however it will be appreciated that the particular connection means isnot critical, and a wireless connection may be used, if desired.

FIG. 3 depicts another preferred configuration of the imaging system.The sample holder 41 contains the sample to be imaged, and smallparticles are present in the sample. The precise structure of the sampleholder is not critical, and could include, e.g., a capillary or glassslide. Illumination source 42 is used to illuminate the sample, ifneeded. Reflecting mirror 43 reflects the image of the sample and bendsthe optical path with an angle. Lens 44 is configured to generate theimage of the sample. Image sensor 45 is configured to sense the image ofthe sample generated by lens 44. Image processing unit 46 is configuredto process the image or images of the sample to determine the positionor movement the particles. In this figure, the image processing unit isdepicted as a laptop computer, but one of ordinary skill would recognizethat many other computing devices could be utilized, such as a desktopcomputer, a tablet computer, a smartphone, an embedded processor in anembedded system, etc. Optional cable 47 connects the image sensor 45 andthe image processor 46; however it will be appreciated that theparticular connection means is not critical, and a wireless connectionmay be used, if desired.

A wide variety of lenses may be used, including a single lens, acompound lens comprising several simple lenses, or an objective lens. Asingle lens comprises a single piece of material, while a compound lenscomprises several simple lenses (elements), usually along a common axis.An objective is the typical lens used in a microscope that gathers lightfrom the object being observed and focuses the light rays to produce areal image.

A telecentric lens is a compound lens that has its entrance or exitpupil at infinity. The entrance pupil of a system is the image of theaperture stop as seen from a point on the optic axis in the objectplane. The exit pupil of a system is the image of the aperture stop asseen from a point on the optic axis in the image plane.

An object space telecentric lens is one type of telecentric lens withits entrance pupil at infinity, which is obtained by positioning thestop aperture on the focal plane of the front optical group of thetelecentric lens. An object space telecentric lens makes objects appearto be the same size independent of their location in space, whichremoves the perspective error that makes closer objects appear to belarger than objects farther from the lens.

An image space telecentric lens is one type of telecentric lens with itsexit pupil at infinity, which is obtained by positioning the stopaperture on the focal plane of the back optical group of the telecentriclens. An image space telecentric lens makes objects appear to be thesame size independent of the placement of the sensor plane, which meansthat sensor placement tolerance for cameras is not as important, as asmall shift toward or away from optimal position will not cause adifference in magnification.

A double telecentric lens is one type of telecentric lens with both itsentrance and exit pupils at infinity, which is obtained by positioningthe stop aperture on the focal plane of both the front and back opticalgroups of the telecentric lens. A double telecentric lens has theadvantage of both an object space telecentric lens and an image spacetelecentric lens, as the field of view is unaffected by shifts of theobject position or the sensor position. It has other advantagesincluding lower image distortion, higher accuracy in object edgeposition, and increased depth of field. Depth of field, also calledfocus range or effective focus range, is the distance between thenearest and farthest objects in a scene that appear acceptably sharp inan image.

Bright-field illumination is the simplest of all the microscopyillumination techniques. The most popular bright-filed illumination istransmitted light (i.e., illuminated from below and observed from aboveor illuminated from above and observed from below) and contrast in thesample is caused by absorbance of some of the transmitted light in denseareas of the sample. The typical appearance of a bright-field microscopyimage is a dark sample on a bright background.

Dark-field illumination is a specialized illumination technique thatcapitalizes on oblique illumination to enhance contrast in specimens.The straight light passing through the specimen from oblique anglescannot enter the microscope lens; only the scattered light from thespecimen can be collected by the lens. As a result, a bright image ofthe specimen superimposed onto a dark background is formed.

An LED ring light is an assembly of LED arranged in a ring style, whichprojects the illumination light to the sample with an oblique angle,usually between 50° and 90°.

An image sensor converts light into electrons and ultimately constitutesan image.

A CCD (charge-coupled device) sensor is one type of image sensor. Whenlight strikes the chip it is held as a small electrical charge in eachpixel, and the charges of all the pixels are transported across the chipand read at one corner of the array. Then, an analog-to-digitalconverter turns each pixel's value into a digital value. An advantage ofa CCD sensor is its high image quality.

A CMOS (complementary metal-oxide semiconductor) sensor is another typeof image sensor. Each pixel of a CMOS image sensor has its owncharge-to-voltage conversion, and also has several transistorsamplifying and manipulating the charge converted from light. The CMOSapproach is more flexible because each pixel works individually. Anadvantage of a CMOS sensor is its low cost and low power consumption.

The image processor used in this invention can be a computer (forexample, a laptop, desktop, tablet or smartphone) or an embeddedprocessor. An embedded processor is a microprocessor that is used in anembedded system. These processors are usually smaller, consume lesspower, and use a surface mount form factor. The form factor is the size,configuration, or physical arrangement of a computing device. Embeddedprocessors can be divided into two categories: ordinary microprocessorsand microcontrollers. Microcontrollers have more peripherals on thechip. In essence, an embedded processor is a CPU chip used in a systemwhich is not a general-purpose workstation, laptop or desktop computer.

The position of a particle in an image can be found by different imageanalysis procedures. In some embodiments of the present invention, theposition of the particle corresponds to the center of the particle thatis found by fitting a two dimensional Gaussian to the particle image. Inother embodiments, the position of the particle corresponds to thecenter of the particle that is found by first generating a binary imageand then finding the geometric center of the binary image of theparticle. A binary image is generated from a grayscale image byassigning a value of one to pixels with intensity higher than a certainthreshold, and a value zero to pixels with intensity lower than thethreshold.

Example

This example demonstrates detection of a 60 nucleotide (nt) syntheticDNA oligonucleotide target (51 in FIG. 4A) having the sequence of asection of Mycobacterium tuberculosis rRNA. The first probe (52 in FIG.4A) was purchased from IDT and consisted of a 24 nt single strandedoligonucleotide having a sequence complementary to the 3′ end of thetarget and a 5′ biotin modification. The second probe (53 in FIG. 4A)was generated in the following manner: A plasmid (8.5 kbps) waslinearized using the restriction enzyme BsmB I (New England Biolabs),which cut the plasmid twice generating a large fragment (˜8.4 kbps˜2.8microns) with different 4 nt overhangs at each end, and a small fragmentwhich was separated and discarded by agarose gel purification (QIAquickGel Extraction Kit, Qiagen). The linearized plasmid was ligated using T4ligase to two double stranded DNA fragments generated by hybridizingsynthetic oligonucleotides. The first fragment had one end with anoverhang compatible to one of the overhangs of the plasmid and the otherend had a 30 nt overhang complementary to the 5′ end of the target. Thesecond fragment had one end with an overhang compatible to the otheroverhang of the plasmid, and the other end of the fragment had a 5′digoxigenin modification. The first probe was mixed with a solutioncontaining the target and the temperature was raised to 65° C. for 1minute and then incubated at room temperature for 10 minutes. The buffercontained 800 mM NaCl. A blocker oligonucleotide complementary to thefirst probe was added and incubated for 5 minutes. The second probe wasadded and incubated for 10 minutes. Super-paramagnetic beads (LifeTechnologies, 54 in FIG. 4A) coated with Streptavidin were added andincubated for 30 minutes. The mixture was then flowed into a glasscapillary tube (5 mm×4 mm×0.2 mm) previously functionalized withanti-digoxigenin proteins (Roche, 55 in FIG. 4A) and beads were letsediment for 5 minutes. A 100 mM NaCl buffer solution was flowed to washunbound beads. The beads were imaged first with flow in one direction(350 microliters/minute, 10 images), and then with flow in the oppositedirection (350 microliters/minute, 10 images), as shown in FIG. 4A. Theoptical system used to image the beads comprised an LED ring light, atelecentric lens and a camera. The LED ring light provided a dark fieldillumination. The telecentric lens exhibited the same magnification forall the beads in the depth of field around 1 millimeter. Themagnification of the system was 1:1, which helped to achieve a largefield of view of 6.14 mm by 4.6 mm. According to the optical resolutionof the lens, the image size of each particle was ˜4 microns, which wassufficient to investigate the displacement of the particles. The cameraused in the system had a 1/2.3 inch (6.14 mm by 4.6 mm) complementarymetal oxide semiconductor (CMOS) chip, which had 4384 by 3288 pixelswith pixel size of 1.4 microns.

A custom code written in Matlab was used to analyze the images anddetermine the position and then displacement of most of the beadspresent in the field of view. The raw image of each particle wastransformed to a binary image in which pixels with intensity higher thana certain threshold were assigned a value of one and pixels withintensity lower than the threshold were assigned a value of zero. Theposition of the particle was then calculated as the center of mass ofthe binary image of the particle. Beads that were too close to eachother (less than about 6 microns) were not included in the analysis. Theposition of a bead was defined as the average of its position in the 10images. This system allowed measurement of the displacement of over10,000 beads in each experiment with sub-micron resolution. The codegenerated a histogram of bead displacement. FIG. 4B shows the detectionof concentrations of 400 femtomolar, 100 femtomolar, and 0 femtomolar ofthe target, respectively. Beads that moved more than 5.8 microns wereconsidered bound to a target-probe complex (right peak in the histogram,57 in FIG. 4B), and the total number of these beads reflects theconcentration of the target. Beads that moved less than 5.8 microns wereconsidered non-specifically attached to the capillary surface (left peakin the histogram, 58 in FIG. 4B).

What is claimed is:
 1. An imaging system, comprising: an illuminationsource configured to illuminate a sample including a target analyte,wherein the target analyte forms a target-probe complex that binds to aparticle on a first end and to a substrate on another end; a lensconfigured to generate a blurred image of the particle, wherein adimension of the blurred image of the particle is greater than adimension of the particle multiplied by the magnification of the lens,and wherein a field of view in an image plane of the lens is larger thantwice a size of the particle; an image sensor having a pixel size ofless than about the dimension of the blurred image of the particle, theimage sensor configured to sense the blurred image of the particle,wherein an intensity of the blurred image of the particle is distributedon at least two pixels to form an intensity distribution; and an imageprocessor coupled with the image sensor and configured to determine acenter of the intensity distribution associated with a first position ofthe particle, wherein the image processor is further configured todetermine a displacement from the first position of the particle to asecond position of the particle in a second configuration of the sample,the displacement being indicative of a binding of the particle to thetarget-probe complex.
 2. The imaging system of claim 1, furthercomprising a sample holder configured to hold a sample containing aparticle, wherein the sample holder is present and comprises a capillaryor a glass slide.
 3. The imaging system of claim 1, wherein: the secondconfiguration of the sample comprises a shifting fluid flow, and theparticle is tethered to the substrate via the target-probe complex, thetarget-probe complex has a pre-selected length, and the image processorverifies the displacement based on the shifting fluid flow and thepre-selected length.
 4. The imaging system of claim 1, wherein the lenshas an optical resolution that is broader than the pixel size in theimage sensor.
 5. The imaging system of claim 1, wherein the imageprocessor is configured to discount the first position of the particlewhen a second particle in the field of view has a position closer to thefirst position of the particle than a resolving power of the lens. 6.The imaging system of claim 1, wherein there are from about 100particles to about 1 million particles in the field of view of thesample.
 7. The imaging system of claim 1, wherein the illuminationsource is present and provides a bright-field illumination or adark-field illumination.
 8. The imaging system of claim 1, wherein thelens comprises a single lens, or a compound lens comprising severalsimple lenses, or an objective lens.
 9. The imaging system of claim 1,wherein the lens comprises a telecentric lens having a magnificationratio of about between 1:5 and 5:1.
 10. The imaging system of claim 1,wherein the image sensor comprises a CCD chip or a CMOS chip.
 11. Amethod of determining a position or a movement of a particle in asample, comprising the steps of: generating a blurred image of theparticle, wherein a dimension of the blurred image of the particle isgreater than the dimension of the particle multiplied by themagnification of the lens, and wherein a field of view in an image planeof the lens is larger than twice a size of the particle; sensing theimage of the particle on at least two pixels of an image sensor having apixel size of less than about the dimension of the image of the particlewherein an intensity of the image of the particle is distributed on atleast two pixels to form an intensity distribution; and processing theimage of the sample, including: determining a center of the intensitydistribution associated with a first position of the particle, anddetermining a displacement from the first position of the particle to asecond position of the particle in a second configuration of the sample,the displacement being indicative of a binding of the particle to atarget-probe complex.
 12. The method of claim 11, further comprisingholding the sample in a sample holder, wherein the sample holdercomprises a capillary or a glass slide.
 13. The method of claim 11,further comprising shifting a fluid flow of the sample to form thesecond configuration of the sample, and verifying the displacement basedon the fluid flow and a pre-selected length of the target-probe complex.14. The method of claim 11, wherein generating a blurred image of theparticle comprises imaging the particle with a lens that has an opticalresolution that is broader than the pixel size in the image sensor. 15.The method of claim 11, further comprising discounting the firstposition of the particle when a second particle in the field of view hasa position closer to the first position of the particle than a resolvingpower of the lens.
 16. The method of claim 11, further comprisingadjusting the field of view of the lens to include from about 100particles to about 1 million particles.
 17. The method of claim 11,further comprising illuminating the sample with a bright-fieldillumination or a dark-field illumination.
 18. The method of claim 11,further comprising illuminating the sample with at least one lightemitting diode.
 19. The method of claim 11, further comprising fitting aGaussian mode to an intensity profile of the image of the particle tofind the center of the particle.
 20. The method of claim 11, furthercomprising finding a geometric center of a binary image of the particleto determine the center of the particle.